The World Health Organization has targeted measles for worldwide eradication, requiring an immunogenic vaccine for the genetically heterogeneous outbred population. Although well controlled by vaccination programs in industrialized countries, measles virus (MV) infection continues to be one of the major causes of childhood morbidity and mortality in developing countries The requirement for a cold chain (storage), the induction of low seroconversion rates in the presence of maternal antibodies, the vaccine failure rate, and the inability to use the vaccine in immuno-compromised persons are the major drawbacks of the live attenuated measles vaccine (El Kasmi, et al., J. Gen. Virol. 81:729-735, 2000; Albrecht, et al., J. Pediatr. 91:715-718, 1977). The limitations of the live vaccine combined with inadequate coverage in developing countries leads to approximately one million measles-related deaths annually (Jaye, et al., J. Clin. Invest. 102:1969-1977, 1998; Sabin, Eur. J. Epidemiol. 7:1-22, 1991). Thus, there is a need to develop alternative vaccines that are thermostable, safe and designed to avoid recognition and neutralization by passive maternal antibodies (El Kasmi, et al., Vaccine 17:2436-2445, 1999; Jaye, et al., supra, 1998). Such vaccines should induce long lasting cell-mediated and humoral immune responses. For this reason, the development of a candidate peptide-based vaccine, based on immunologically relevant information on naturally processed and presented measles virus-derived peptides eluted from HLA class I and class II antigen-presenting molecules, would have a significant impact.
Defining peptide epitopes recognized by CD8+ and CD4+ T lymphocytes involved in immune responses has generated tremendous interest (Germain, Cell 76:287-299, 1994a). We previously demonstrated that humoral immune responses to measles-encoded proteins are strongly associated with the human leukocyte antigen (HLA) class I and class II genes (Poland, et al., Vaccine 17:1719-1725, 1999). In particular, HLA-DRB1*03 (DR3) alleles are significantly associated with measles vaccine seronegativity and play an important role in the immune response to MV (Poland, et al., Vaccine 20:430-438, 2001a). Identification and comparison of the repertoire of measles-derived peptides that bind to class II HLA-DR3 molecules in poor- and high-responders to measles vaccine is important for design of more effective vaccines against measles. The HLA class I and class II antigen-processing pathways play a critical role in the activation of measles-specific T-lymphocytes by presenting peptide epitopes derived from viral proteins (Pamer, Clin. Infect. Dis. 28:714-716, 1999). The HLA class II molecules bind and present exogenous measles antigens for recognition by CD4+ T-helper cells and play an important role in the immune response to measles (Germain, Int. J. Technol. Assess. Health Care 10:81-92, 1994b; Germain, Ann. NY Acad. Sci. 754:114-125, 1995; Pamer, supra, 1999). Alternatively, class II molecules can also use the endogenous pathway of measles virus antigen presentation (Nuchtem, et al., Nature 343:74-76, 1990; Sekaly, et al., Proc. Natl. Acad. Sci. USA 85:1209-1212, 1988). Identification of such immunogenic measles epitopes, which are recognized by T- and B-lymphocytes would advance peptide-base therapies and vaccine development (Poland, et al., Vaccine 19:2692-2700, 2001b). However, a potential obstacle to the development of a peptide-based measles vaccine is the high degree of human HLA gene polymorphism (Doolan, et al., J. Immunol. 1123-1137, 2000).
HLA molecules bind antigenic peptides and display them to T cell receptors on the surface of helper T cells (Garcia, et al., Annu. Rev. Immunol. 17:369-397, 1999; Brown, et al., Nature 332:845-850, 1988; Stern, et al., Nature 368:215-221, 1994). Adoptive immune responses are therefore limited by the spectrum of immunogenic peptides displayed to T cells. Limitations in identifying class II peptides include the difficulty in detecting pathogen-derived peptides eluted from HLA class II-peptide complexes and the lack of knowledge regarding HLA class II presentation of measles virus peptides, as only a few human measles virus class I peptides and HLA class II-restricted cytotoxic T lymphocytes (CTL) responses are described in the literature (Herberts, et al., J. Gen. Virol. 82:2131-2142, 2001; van Els, et al., Eur. J. Immunol. 30:1172-1181, 2000; van Binnendijk, et al., J. Virol. 67:2276-2284, 1993; Jacobson, et al., J. Virol. 63:1756-1762, 1989). However, the rapid characterization of defined peptides that are critical to viral immunity, including measles, has been significantly enhanced by mass spectrometry (MS), which provides peptide sequence information at the femtomole level of sensitivity.
Although direct sequencing of naturally processed peptides bound to HLA Class I and II molecules by liquid chromatography mass spectrometry (LC-MS) is established (Dongre, et al., Eur. J. Immunol. 31:1485-1494, 2001; de Jong, Mass Spectrom. Rev. 17:311-335, 1998), identification of pathogen-derived peptides presents a formidable challenge due to the diverse range of low abundance peptides presented by HLA molecules. Strategies to reduce the complexity of the mixture prior to introduction into the mass spectrometer have often relied on multiple steps of reversed phase (RP) liquid chromatography. However, this approach does not effectively increase the peak capacity because the separation mechanisms of each RP chromatography step are not orthogonal.
Needed in the art of measles diagnosis and vaccine development are diagnostic and therapeutic methods that depend on the isolation of naturally processed measles virus peptides eluted from class II HLA molecules.